High-throughput synthesis of stable isotope-labeled transmembrane proteins for targeted transmembrane proteomics using a wheat germ cell-free protein synthesis system

Takemori, Nobuaki; Takemori, Ayako; Matsuoka, Katsunari; Morishita, Ryo; Matsushita, Natsuki; Aoshima, Masato; Takeda, Hiroyuki; Sawasaki, Tatsuya; Endo, Yaeta; Higashiyama, Shigeki

doi:10.1039/c4mb00556b

Cited by 23 publications

(28 citation statements)

References 23 publications

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“…Since PNPLA1 is a membrane protein32, we added liposomes to the translation reaction mixture. Recently, several membrane proteins have successfully been inserted directly into the lipid bilayer of liposomes by similar cell-free translation systems333435. After translation of PNPLA1 mRNA, the resulting proteoliposomes were recovered by centrifugation and used for further analyses.…”

Section: Resultsmentioning

confidence: 99%

PNPLA1 is a transacylase essential for the generation of the skin barrier lipid ω-O-acylceramide

et al. 2017

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Lipids are the primary components of the skin permeability barrier, which is the body's most powerful defensive mechanism against pathogens. Acylceramide (ω-O-acylceramide) is a specialized lipid essential for skin barrier formation. Here, we identify PNPLA1 as the long-sought gene involved in the final step of acylceramide synthesis, esterification of ω-hydroxyceramide with linoleic acid, by cell-based assays. We show that increasing triglyceride levels by overproduction of the diacylglycerol acyltransferase DGAT2 stimulates acylceramide production, suggesting that triglyceride may act as a linoleic acid donor. Indeed, the in vitro analyses confirm that PNPLA1 catalyses acylceramide synthesis using triglyceride as a substrate. Mutant forms of PNPLA1 found in patients with ichthyosis exhibit reduced or no enzyme activity in either cell-based or in vitro assays. Altogether, our results indicate that PNPLA1 is directly involved in acylceramide synthesis as a transacylase, and provide important insights into the molecular mechanisms of skin barrier formation and of ichthyosis pathogenesis.

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Section: Resultsmentioning

confidence: 99%

PNPLA1 is a transacylase essential for the generation of the skin barrier lipid ω-O-acylceramide

et al. 2017

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“…Cell-free system is free from the control and effect of intracellular trafficking and signaling pathways, and thus it has the potential to produce variety of membrane proteins efficiently 15 . Indeed, reports have increased much lately on cell-free synthesis of membrane proteins, in which liposome or detergent is added into the reaction 15 16 17 18 19 20 21 . However, it is not practical enough to prepare mg of immunizing antigen using the existing cell-free methods considering their limited productivity.…”

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confidence: 99%

Production of monoclonal antibodies against GPCR using cell-free synthesized GPCR antigen and biotinylated liposome-based interaction assay

Takeda

Ogasawara

Ozawa

et al. 2015

Sci Rep

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G-protein-coupled receptors (GPCRs) are one of the most important drug targets, and anti-GPCR monoclonal antibody (mAb) is an essential tool for functional analysis of GPCRs. However, it is very difficult to develop GPCR-specific mAbs due to difficulties in production of recombinant GPCR antigens, and lack of efficient mAb screening method. Here we describe a novel approach for the production of mAbs against GPCR using two original methods, bilayer-dialysis method and biotinylated liposome-based interaction assay (BiLIA), both of which are developed using wheat cell-free protein synthesis system and liposome technology. Using bilayer-dialysis method, various GPCRs were successfully synthesized with quality and quantity sufficient for immunization. For selection of specific mAb, we designed BiLIA that detects interaction between antibody and membrane protein on liposome. BiLIA prevented denaturation of GPCR, and then preferably selected conformation-sensitive antibodies. Using this approach, we successfully obtained mAbs against DRD1, GHSR, PTGER1 and T1R1. With respect to DRD1 mAb, 36 mouse mAbs and 6 rabbit mAbs were obtained which specifically recognized native DRD1 with high affinity. Among them, half of the mAbs were conformation-sensitive mAb, and two mAbs recognized extracellular loop 2 of DRD1. These results indicated that this approach is useful for GPCR mAb production.

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“…To resolve this ambiguity, we performed an in vitro FA ␣-oxidation assay using purified 3ϫFLAG-Mpo1 translated by a wheat germ cell-free translation system in the presence of liposomes. This method reportedly inserts membrane proteins directly into the lipid bilayer of liposomes (26)(27)(28)(29). After cell-free translation of 3ϫFLAG-Mpo1, the resulting proteoliposomes were separated from the wheat germ lysates by centrifugation.…”

Section: Resultsmentioning

confidence: 99%

Yeast Mpo1 Is a Novel Dioxygenase That Catalyzes the α-Oxidation of a 2-Hydroxy Fatty Acid in an Fe²⁺-Dependent Manner

Seki

Mori

Kitamura

et al. 2019

Molecular and Cellular Biology

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Phytosphingosine (PHS) is the major long-chain base component of sphingolipids in Saccharomyces cerevisiae. The PHS metabolic pathway includes a fatty acid (FA) α-oxidation reaction. Recently, we identified the novel protein Mpo1, which is involved in PHS metabolism. However, the details of the FA α-oxidation reaction and the role of Mpo1 in PHS metabolism remained unclear. In the present study, we revealed that Mpo1 is involved in the α-oxidation of 2-hydroxy (2-OH) palmitic acid (C16:0-COOH) in the PHS metabolic pathway. Our in vitro assay revealed that not only the Mpo1-containing membrane fraction but also the soluble fraction was required for the α-oxidation of 2-OH C16:0-COOH. The addition of Fe2+ eliminated the need for the soluble fraction. Purified Mpo1 converted 2-OH C16:0-COOH to C15:0-COOH in the presence of Fe2+, indicating that Mpo1 is the enzyme body responsible for catalyzing the FA α-oxidation reaction. This reaction was also found to require an oxygen molecule. Our findings indicate that Mpo1 catalyzes the FA α-oxidation reaction as 2-OH fatty acid dioxygenase, mediated by iron(IV) peroxide. Although numerous Mpo1 homologs exist in bacteria, fungi, protozoa, and plants, their functions had not yet been clarified. However, our findings suggest that these family members function as dioxygenases.

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High-throughput synthesis of stable isotope-labeled transmembrane proteins for targeted transmembrane proteomics using a wheat germ cell-free protein synthesis system

Cited by 23 publications

References 23 publications

PNPLA1 is a transacylase essential for the generation of the skin barrier lipid ω-O-acylceramide

PNPLA1 is a transacylase essential for the generation of the skin barrier lipid ω-O-acylceramide

Production of monoclonal antibodies against GPCR using cell-free synthesized GPCR antigen and biotinylated liposome-based interaction assay

Yeast Mpo1 Is a Novel Dioxygenase That Catalyzes the α-Oxidation of a 2-Hydroxy Fatty Acid in an Fe²⁺-Dependent Manner

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High-throughput synthesis of stable isotope-labeled transmembrane proteins for targeted transmembrane proteomics using a wheat germ cell-free protein synthesis system

Cited by 23 publications

References 23 publications

PNPLA1 is a transacylase essential for the generation of the skin barrier lipid ω-O-acylceramide

PNPLA1 is a transacylase essential for the generation of the skin barrier lipid ω-O-acylceramide

Production of monoclonal antibodies against GPCR using cell-free synthesized GPCR antigen and biotinylated liposome-based interaction assay

Yeast Mpo1 Is a Novel Dioxygenase That Catalyzes the α-Oxidation of a 2-Hydroxy Fatty Acid in an Fe2+-Dependent Manner

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Yeast Mpo1 Is a Novel Dioxygenase That Catalyzes the α-Oxidation of a 2-Hydroxy Fatty Acid in an Fe²⁺-Dependent Manner